Zinc-free and low-zinc insulin preparations having improved stability

ABSTRACT

The invention relates to a formulation comprising a polypeptide selected from at least one of insulin, an insulin metabolite, an insulin analog, and an insulin derivative; at least one surfactant; optionally at least one preservative; and optionally at least one of an isotonicizing agent, a buffer or an excipient, wherein the formulation is free from or low in zinc. The invention also relates to the production of such insulin preparations and their use as pharmaceutical formulations.

This application is a division of application Ser. No. 10/102,862, filedMar. 22, 2002, now U.S. Pat. No. 6,960,561, and claims priority toGerman Application No. 101 14 178.5, filed Mar. 23, 2001, each of whichare incorporated by reference herein.

This application claims the priority of German Patent Application No.10114178.5, filed Mar. 23, 2001, which is incorporated herein byreference.

The invention relates to stabilized pharmaceutical formulationscomprising a polypeptide selected from at least one of insulin (e.g.human insulin, bovine insulin, or porcine insulin, or another animalinsulin), an insulin analog, an insulin derivative, and active insulinmetabolites, or combinations thereof; at least one surfactant, orcombinations of a number of surfactants; optionally at least onepreservative, or combinations of a number of preservatives; andoptionally at least one of an isotonicizing agent, a buffer or anexcipient, or combinations thereof, wherein the pharmaceuticalformulation is low in zinc or free from zinc. These formulations can beemployed as pharmaceutical or other medicinal formulations, for example,for the treatment of diabetes. They are particularly employable for usein insulin pumps, pens, injectors, inhalers, or for any use in whichincreased physical stability of the preparation is necessary. Theinvention likewise relates to parenteral preparations which contain suchformulations and can be used in diabetes. The invention also relates tomethods for producing the preparations and to methods for improving thestability of insulin preparations.

Worldwide, approximately 120 million people suffer from diabetesmellitus. Among these, approximately 12 million are type I diabetics,for whom the substitution of the lacking endocrine insulin secretion isthe only currently possible therapy. The affected persons are dependentlifelong on insulin injections, as a rule a number of times daily. Incontrast to type I diabetes, there is not basically a deficiency ofinsulin in type II diabetes, but in a large number of cases, especiallyin the advanced stage, treatment with insulin, optionally in combinationwith an oral antidiabetic, is regarded as the most favorable form oftherapy.

In the healthy person, the release of insulin by the pancreas isstrictly coupled to the concentration of the blood glucose. Elevatedblood glucose levels, such as occur after meals, are rapidly compensatedby a corresponding increase in insulin secretion. In the fasting state,the plasma insulin level falls to a basal value which is adequate toguarantee a continuous supply of insulin-sensitive organs and tissuewith glucose and to keep hepatic glucose production low in the night.The replacement of the endogenous insulin secretion by exogenous, mostlysubcutaneous administration of insulin as a rule does not approximatelyachieve the quality of the physiological regulation of the blood glucosedescribed above. Often, deviations of the blood glucose upward ordownward occur, which in their severest forms can be life-threatening.In addition, however, blood glucose levels which are increased for yearswithout initial symptoms are a considerable health risk. The large-scaleDCCT study in the USA (The Diabetes Control and Complications TrialResearch Group (1993) N. Engl. J. Med. 329, 977–986) demonstratedclearly that chronically elevated blood glucose levels are essentiallyresponsible for the development of diabetic late damage. Diabetic latedamage is microvascular and macrovascular damage which is manifested,under certain circumstances, as retinopathy, nephropathy or neuropathyand leads to loss of sight, kidney failure and the loss of extremitiesand is moreover accompanied by an increased risk of cardiovasculardiseases. It is to be derived from this that an improved therapy ofdiabetes is primarily to be aimed at keeping the blood glucose asclosely as possible in the physiological range. According to the conceptof intensified insulin therapy, this should be achieved by repeateddaily injections of rapid- and slow-acting insulin preparations.Rapid-acting formulations are given at meals in order to level out thepostprandial increase in the blood glucose. Slow-acting basal insulinsshould ensure the basic supply with insulin, in particular during thenight, without leading to hypoglycemia.

Insulin is a polypeptide of 51 amino acids, which are divided into 2amino acid chains: the A chain having 21 amino acids and the B chainhaving 30 amino acids. The chains are connected to one another by meansof 2 disulfide bridges. Insulin preparations have been employed fordiabetes therapy for many years. Not only naturally occurring insulinsare used here, but recently also insulin derivatives and analogs.

Insulin analogs are analogs of naturally occurring insulins, namelyhuman insulin or animal insulins, which differ by substitution of atleast one naturally occurring amino acid residue with other amino acidresidues and/or addition/removal of at least one amino acid residue fromthe corresponding, otherwise identical, naturally occurring insulin. Theadded and/or replaced amino acid residues can also be those which do notoccur naturally.

Insulin derivatives are derivatives of naturally occurring insulin or ofan insulin analog which are obtained by chemical modification. Thechemical modification can consist, for example, in the addition,substitution or deletion of one or more specific chemical groups to oneor more amino acids. It can also involve the addition, substitution ordeletion of one or more chemical groups of the peptide backbone, suchas, at the amino and/or carboxyl terminus.

Active insulin metabolites of naturally occurring insulin, of insulinanalogs, or of insulin derivatives may be formed in the formulations ofthe invention by chemical, enzymatic, or oxidative means. Active insulinmetabolites retain at least partial insulin activity. Examples areproducts of chemical or oxidative degradation of insulin polypeptides.

As a rule, insulin derivatives and insulin analogs have a somewhatmodified action compared with human insulin.

Insulin analogs having an accelerated onset of action are described inEP 0 214 826, EP 0 375 437 and EP 0 678 522. EP 0 124 826 relates, interalia, to substitutions of B27 and B28. EP 0 678 522 describes insulinanalogs which have various amino acids, preferably proline, in positionB29, but not glutamic acid.

EP 0 375 437 includes insulin analogs with lysine or arginine in B28,which can optionally additionally be modified in B3 and/or A21.

In EP 0 419 504, insulin analogs are disclosed which are protectedagainst chemical modifications, in which asparagine in B3 and at leastone further amino acid in the positions A5, A15, A18 or A21 aremodified.

In WO 92/00321, insulin analogs are described in which at least oneamino acid of the positions B1-B6 is replaced by lysine or arginine.According to WO 92/00321, insulins of this type have a prolonged action.

The insulin preparations of naturally occurring insulins on the marketfor insulin substitution differ in the origin of the insulin (e.g.bovine, porcine, human insulin, or another mammalian or animal insulin),and also the composition, whereby the profile of action (onset of actionand duration of action) can be influenced. By combination of variousinsulin preparations, very different profiles of action can be obtainedand blood sugar values which are as physiological as possible can beestablished. Preparations of naturally occurring insulins, as well aspreparations of insulin derivatives or insulin analogs which showmodified kinetics, have been on the market for some time. RecombinantDNA technology today makes possible the preparation of such modifiedinsulins. These include “monomeric insulin analogs” such as insulinLispro, insulin Aspart, and HMR 1964 (Lys(B3), Glu(B29) human insulin),all of which have a rapid onset of action, as well as insulin Glargin,which has a prolonged duration of action.

In addition to the duration of action, the stability of the preparationis very important for patients. Stabilized insulin formulations havingincreased physical long-term stability are needed in particular forpreparations which are exposed to particular mechanical stresses orrelatively high temperatures. These include, for example, insulins inadministration systems such as pens, inhalation systems, needlelessinjection systems or insulin pumps. Insulin pumps are either worn on orimplanted in the body of the patient. In both cases, the preparation isexposed to the heat of the body and movement and to the delivery motionof the pump and thus to a very high thermomechanical stress. Sinceinsulin pens too (disposable and reutilizable pens) are usually worn onthe body, the same applies here. Previous preparations have only alimited stability under these conditions.

Insulin is generally present in neutral solution in pharmaceuticalconcentration in the form of stabilized zinc-containing hexamers, whichare composed of 3 identical dimer units (Brange et al., Diabetes Care13:923–954 (1990)). However, the profile of action an insulinpreparation may be improved by reducing the oligomeric state of theinsulin it contains. By modification of the amino acid sequence, theself-association of insulin can be decreased. Thus, the insulin analogLispro, for example, mainly exists as a monomer and is thereby absorbedmore rapidly and shows a shorter duration of action (HPT Ammon and C.Werning; Antidiabetika [Antidiabetics]; 2. Ed.; Wiss. Verl.-Ges.Stuttgart; 2000; p. 94.f). However, the rapid-acting insulin analogswhich often exist in the monomeric or dimeric form are less stable andmore prone to aggregate under thermal and mechanical stress thanhexameric insulin. This makes itself noticeable in cloudiness andprecipitates of insoluble aggregates. (Bakaysa et al, U.S. Pat. No.5,474,978). These higher molecular weight transformation products(dimers, trimers, polymers) and aggregates decrease not only the dose ofinsulin administered but can also induce irritation or immune reactionsin patients. Moreover, such insoluble aggregates can affect and blockthe cannulas and tubing of the pumps or needles of pens. Since zincleads to an additional stabilization of insulin through the formation ofzinc-containing hexamers, zinc-free or low-zinc preparations of insulinand insulin analogs are particularly susceptible to instability. Inparticular, monomeric insulin analogs having a rapid onset of action areprone to aggregate and become physically unstable very rapidly, becausethe formation of insoluble aggregates proceeds via monomers of insulin.

In order to guarantee the quality of an insulin preparation, it isnecessary to avoid the formation of aggregates. There are variousapproaches for stabilizing insulin formulations. Thus, in internationalpatent application WO98/56406, formulations stabilized by TRIS orarginine buffer have been described. U.S. Pat. No. 5,866,538 describesan insulin preparation which contains glycerol and sodium chloride inconcentrations of 5–100 mM and should have an increased stability. U.S.Pat. No. 5,948,751 describes insulin preparations having increasedphysical stability, which is achieved by addition of mannitol or similarsugars. The addition of excess zinc to a zinc-containing insulinsolution can likewise increase the stability (J. Brange et al., DiabeticMedicine, 3: 532–536, 1986). The influence of the pH and variousexcipients on the stability of insulin preparations has also beendescribed in detail (J. Brange & L. Langkjaer, Acta Pharm. Nordica 4:149–158).

Often, these stabilization methods are not adequate for increaseddemands (improvement in ability to be kept at room or body temperatureand under mechanical stress) or for “monomeric” insulin analogs orrapid-acting insulins, which are particularly susceptible to physicalstress. Moreover, all commercial insulin preparations contain zinc,which is added to stabilize the preparation. Thus, Bakaysa et al. inU.S. Pat. No. 5,474,978 describe stabilized formulations of insulincomplexes which consist of 6 insulin analog monomers, 2 zinc atoms andat least 3 molecules of a phenolic preservative. These formulations canadditionally contain a physiologically acceptable buffer and apreservative. If it is wished, however, to prepare zinc-free or low-zincinsulin preparations, the stabilization methods mentioned are notadequate for a marketable preparation. For example, it was not possibleto develop a zinc-free preparation of insulin Lispro on account ofinadequate physical stability (Bakaysa et al., Protein Science (1996),5:2521–2531). Low-zinc or zinc-free insulin formulations having adequatestability, in particular physical stability, are not described in theprior art.

The present invention was thus based on the object of finding zinc-freepreparations for insulins and their derivatives and analogs, which aredistinguished by a high stability.

It has now surprisingly been found that the addition of surfactants(emulsifiers) such as, for example, poloxamers or polysorbates (such aspolyoxyethylenesorbitan monolaurate (TWEEN® 20) can drastically increasethe stability of insulin preparations. Thus, even zinc-free preparationscan be prepared which have a superior stability, and which are capableof being used in infusion pumps or other administration systems. Thesepreparations show increased stability, particularly under stressconditions. This finding applies to insulin, insulin analogs, andinsulin derivatives, as well as to mixtures of insulin, insulin analogs,and insulin derivatives.

In neutral preparations, insulin forms complexes with zinc ions. Here,at an adequate zinc concentration, stable hexamers are formed from 6insulin molecules and 2 zinc ions. For the formation of this structure,a zinc concentration of at least 0.4% (w/w) relative to the insulin isnecessary. This corresponds in the case of a preparation of 100 IU/ml ofinsulin to a concentration of about 13 μg/ml of zinc. An excess of zinc(e.g. 4 zinc ions per hexamer) again markedly stabilizes the preparationagainst physical stress (J. Brange et al., Neutral insulin solutionsphysically stabilized by the addition of Zn²⁺. Diabetic Med. 3, 532–536(1986)). In contrast to this, in preparations having lower zincconcentrations (<0.4 percent by weight based on insulin), the formationof the hexamers is reduced. This leads to a dramatically reducedstability of the preparation (J. Brange and L. Langkjaer; Acta PharmNord, 4: 149–158 (1992)). “Zinc-free” or “low-zinc” within the meaningof this application therefore means the presence of less than 0.4percent by weight of zinc based on the insulin content of thepreparation, for example, less than 0.2 percent by weight based on theinsulin content. For a customary insulin preparation containing 100units per milliliter (0.6 μmol/ml), this means, for example, aconcentration of less than 13 μg/ml of Zn⁺⁺ ions (0.2 μmol/ml), forexample, less than 6.5 μg/ml of Zn⁺⁺ ions, in the pharmaceuticalpreparation, based on an insulin concentration of 100 units/ml. Thefreedom from zinc can also be achieved by addition of zinc-complexingsubstances, such as, for example, citrate or EDTA, so that sufficientzinc ions are not available for the formation of the insulin/zinchexamer complex.

The pharmaceutical preparations contain about 60–6000 nmol/ml, forexample, about 240–3000 nmol/ml, of an insulin, an active insulinmetabolite, an insulin analog or an insulin derivative.

Surfactants which can be used are, inter alia, nonionic or ionic(anionic, cationic or amphoteric) surfactants. Pharmaceuticallycustomary surfactants may be used, such as: alkali metal soaps, aminesoaps and alkaline earth metal soaps (for example, stearates,palmitates, oleates, and ricinoleates), alkylsulfates andalkylsulfonates (for example, sodium laurylsulfate, sodium cetylsulfate,and sodium stearylsulfate), natural surfactants (such as, bile acidsalts, saponins, and gum arabic), cationic surfactants (such as,alkonium bromides, cetylpyridinium chloride, and cetrimide), fattyalcohols (for example, cetyl alcohol, stearyl alcohol, and cholesterol),esters, such as fatty acid esters, and ethers of polyhydric alcohols(e.g. esters or ethers of glycerol, sorbitol, polyethylene glycol andthe like (e.g. SPAN®, TWEEN®, MYRJ®, BRIJ®, and CREMOPHOR®), and polyols(e.g. poloxamers).

The surfactants may be present in the pharmaceutical composition in aconcentration of about 0.1 μg/ml–10000 μg/ml, for example, about 1μg/ml–1000 μg/ml.

The preparation can additionally contain preservatives (e.g. phenol,cresol, and parabens), isotonicizing agents (e.g. mannitol, sorbitol,lactose, dextrose, trehalose, sodium chloride, and glycerol), buffersubstances, salts, acids and alkalis, and further excipients. Thesesubstances can in each case be present individually or alternatively asmixtures.

Glycerol, dextrose, lactose, sorbitol and mannitol are customarilypresent in the pharmaceutical preparation in a concentration of about100–250 mM. NaCl may be present in a concentration of up to about 150mM. Buffer substances, such as, for example, phosphate, acetate,citrate, arginine, glycylglycine or TRIS (i.e.2-amino-2-hydroxymethyl-1,3-propanediol) buffer and corresponding salts,may be present in a concentration of about 5–250 mM, generally about10–100 mM. Further excipients can, inter alia, be salts, arginine,protamine, or SURFEN® (also referred to herein as amino quinurid (INN),or 1,3-bis(4-amino-2-methyl-6-chinolyl) urea).

The invention therefore relates to a formulation comprising apolypeptide selected from at least one of insulin, an insulin analog, aninsulin derivative, and an active insulin metabolite; at least onesurfactant; optionally at least one preservative; and optionally atleast one of an isotonicizing agent, buffer substances and/or furtherexcipients. The formulation is either free from or low in zinc.

In one embodiment, the surfactant is selected from at least one ofalkali metal soaps, amine soaps, alkaline earth metal soaps,alkylsulfates, alkylsulfonates, natural surfactants, cationicsurfactants, fatty alcohols, partial and fatty acid esters of polyhydricalcohols (such as of glycerol and sorbitol), ethers of polyhydricalcohols, polyethylene glycol ethers, and polyols.

In some embodiments, the alkali metal, amine, and alkaline earth metalsoaps mentioned are selected from at least one of stearates, palmitates,oleates, and ricinoleates. In some embodiments, the alkylsulfates areselected from at least one of sodium laurylsulfate, sodium cetylsulfate,and sodium stearylsulfate.

In some embodiments, the natural surfactants are selected from at leastone of bile acid salts, saponins, gum arabic, and lecithins. Thecationic surfactants may be selected from at least one of alkoniumbromides, cetylpyridinium chloride, and CETRIMIDE® (analkyltrimethylammonium bromide).

In some embodiments, the fatty alcohols are selected from at least oneof cetyl alcohol, stearyl alcohol, and cholesterol.

In some embodiments, esters and ethers of polyhydric alcohols such asglycerol, polyethylene glycol, sucrose, and sorbitol are selected assurfactants. The esters and ethers of polyhydric alcohols include fattyacid esters and ethers. The esters and ethers may also be partial estersand ethers, in which only some hydroxyl groups are modified to the esteror ether form, or they may be complete esters or ethers, in which allhydroxyl groups are modified. Examples of esters and ethers ofpolyhydric alcohols that may be employed in this invention include SPAN®(fatty acid esters of sorbitan), TWEEN® (polysorbates, fatty acid estersof polyoxyethylenesorbitan), MYRJ® (polyoxyethylene stearates), BRIJ®(polyoxyethylene ethers), and/or CREMOPHOR® (polyoxyethylene fatty acidesters), each of which are commercially available, and may be employed,in a variety of molecular weights.

In some embodiments, the surfactant may be a polyol. Example polyols arepolypropylene glycols, polyethylene glycols, poloxamers, Pluronics, andTetronics.

The preservative, in some embodiments, is selected from at least one ofphenol, cresol, and parabens.

In some embodiments, the isotonicizing agent is selected from at leastone of mannitol, sorbitol, sodium chloride, and glycerol. Theexcipients, in some embodiments, are selected from buffer substances,acids, alkalis, salts, protamine, arginine, and SURFEN®.

In some embodiments, the polypeptide of the preparation is an insulinoccurring in nature, for example human, bovine or porcine insulin, orthe insulin of another animal or mammal. In some embodiments, thepolypeptide of the preparation comprises an insulin analog, selectedfrom at least one of Gly(A21)-Arg(B31)-Arg(B32) human insulin;Lys(B3)-Glu(B29) human insulin; Lys^(B28)Pro^(B29) human insulin, B28Asp human insulin, human insulin, in which proline in position B28 hasbeen substituted by Asp, Lys, Leu, Val or Ala and where in position B29Lys can be substituted by Pro; AlaB26 human insulin; des(B28-B30) humaninsulin; des(B27) human insulin or des(B30) human insulin. In additionalembodiments, the polypeptide of the preparation comprises an insulinderivative selected from at least one of B29-N-myristoyl-des(B30) humaninsulin, B29-N-palmitoyl-des(B30) human insulin, B29-N-myristoyl humaninsulin, B29-N-palmitoyl human insulin, B28-N-myristoylLys^(B28)Pro^(B29) human insulin, B28-N-palmitoyl-Lys^(B28)Pro^(B29)human insulin, B30-N-myristoyl-Thr^(B29)Lys^(B30) human insulin,B30-N-palmitoyl-Thr^(B29)Lys^(B30) human insulin,B29-N-(N-palmitoyl-γ-glutamyl)-des (B30) human insulin,B29-N-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin,B29-N-(ω-carboxyheptadecanoyl)-des(B30) human insulin, andB29-N-(ω-carboxyheptadecanoyl) human insulin. In some embodiments, thepolypeptide may comprise an active insulin metabolite. Some embodimentscomprise preparations containing mixtures of one or more of an insulin,an insulin analog, an insulin derivative, and an active insulinmetabolite, for example, selected from those described above.

The invention further relates to a pharmaceutical formulation asdescribed above, in which the insulin, the insulin analog, the activeinsulin metabolite and/or the insulin derivative is present in aconcentration of 60–6000 nmol/ml, such as a concentration of 240–3000nmol/ml (which corresponds approximately to a concentration of 1.4–35mg/ml or 40–500 units/ml). The surfactant may be present in aconcentration of 0.1–10000 μg/ml, such as a concentration of 1–1000μg/ml.

The invention further relates to a pharmaceutical formulation asmentioned above, in which glycerol and/or mannitol is present in aconcentration of 100–250 mM, and/or chloride is present in aconcentration of up to 150 mM.

The invention further relates to a pharmaceutical formulation asmentioned above, in which a buffer substance is present in aconcentration of 5–250 mM.

The invention further relates to a pharmaceutical insulin formulationwhich contains further additives such as, for example, salts, protamine,or SURFEN®, which delay the release of insulin. Mixtures of suchdelayed-release insulins with formulations as described above are alsoincluded herein.

The invention further relates to a method for the preparation of suchpharmaceutical formulations. For example, the components may be mixedtogether in the form of aqueous solutions, after which the pH isadjusted to a desired level, and the mixture is made up to the finalvolume with water. In some embodiments, after the mixture is made up tothe final volume with water the mixture may comprise 1–5 mg/ml ofcresol, for example, about 3 mg/ml, or about 3.15 mg/ml; 1.4–35 mg/ml ofinsulin, an insulin analog, an insulin derivative, and/or an activeinsulin metabolite, such as, 3–5 mg/ml, 3–4 mg/ml, or about 3.5 mg/ml;about 1–10 mg/ml of trometamol, for example, about 5–7 mg/ml, or about6.0 mg/ml of trometamol; 1–8 mg/ml of NaCl, such as, about 4–6 mg/ml, orabout 5.0 mg/ml of NaCl; and 1–1000 μg/ml of TWEEN® 20, for example,about 10–100 μg/ml, or about 100 μg/ml (0.1 mg/ml) of TWEEN® 20. In someembodiments, the polypeptide present in this final mixture comprisesLys(B3), Glu(B29) human insulin (HMR 1964).

The invention further likewise relates to the administration of suchformulations for the treatment of diabetes mellitus. The formulationsmay be administered to mammalian patients, such as humans and domesticmammals.

The invention further relates to the use or the addition of surfactantsas stabilizer during the process for the preparation of insulin, insulinanalogs or insulin derivatives or their preparations.

In the pharmaceutical formulations described, the polypeptide isselected from insulin, an insulin analog, an insulin derivative, and/oran active insulin metabolite, the pH is between 2 and 12, generallybetween 6 and 8.5, and often between 7 and 7.8.

The application is described below with the aid of some examples, whichshould in no case act in a restrictive manner.

EXAMPLES

Comparison investigations: Various zinc-free preparations containing theinsulin analog HMR1964 (Lys(B3), Glu(B29), human insulin) were prepared.To this end, zinc-free HMR1964 and the other constituents were dissolvedin one part of water for injection purposes and the pH was adjusted to7.3+/−0.2 with hydrochloric acid/NaOH and made up to the final volume.The concentration of HMR1964 in each of the experiments described belowwas 3.5 mg/ml (corresponds to 100 units/ml). A second preparation wasprepared identically, but a specific amount of a surfactant wasadditionally added. The solutions were dispensed into 5 ml or 10 mlglass vessels (vials) and fitted with crimp caps. These vessels werethen exposed to the following stress conditions.

1. Rotation test: In each case 5 vessels of a batch and 5 vessels of thecomparison batch were subjected to a rotation test. To this end, thevessels were mounted in a rotator and rotated top over bottom (360°) at37° C. at 60 rpm. After defined times, the turbidity of the preparationssituated in the vessels was compared with turbidity standards ordetermined in formazine nephelometric units (FNU) using a laboratoryturbidity photometer (nephelometer). The experiment was carried outuntil a turbidity value of 18 FNU was exceeded in all vessels.

2. Shaking test: The vessels were placed on a laboratory shaker in anincubator and shaken at 30° C. at 100 movements/min. After definedtimes, the turbidity value of the samples was determined by means of alaboratory turbidity photometer (nephelometer) in formazinenephelometric units (FNU).

Example 1

Stabilization of HMR1964 by Addition of Zinc in the Rotation Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution, which in the final formulation contained 2.7 mg/ml ofm-cresol, 20 mg/ml of glycerol and 6 mg/ml of trometamol (tris), and thepH was adjusted to 7.2–7.4 (measured at room temperature) using 1 Nhydrochloric acid/1 N NaOH. The solution was made up to the final volumewith water and sterile-filtered through a 0.2 μm filter. It was thenfilled into 5 ml injection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength zinc chloride stocksolution was added, so that a zinc content of 15 μg/ml results in thefinished formulation.

In each case, 5 samples were then stressed in the rotation test and theturbidity was determined after various periods of time. The results areshown in the following table.

Number of test samples with turbidity >18 FNU after: Description 0 h 8 h16 h 32 h 40 h 56 h HMR1964 without addition 0 5 — — — — HMR1964 + 15μg/ml of Zn 0 0 0 0 4 5

The addition of zinc markedly delayed the resulting turbidity of thesolution in terms of time and thereby stabilized the HMR1964formulation. Without addition of zinc, the preparation had a markedturbidity in the rotation test even after 8 hours.

Example 2

Stabilization of HMR1964 by Addition of Polysorbate 20 (TWEEN® 20) inthe Rotation Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution which contained 3.15 mg/ml of m-cresol, 5 mg/ml of NaCl and 6mg/ml of trometamol in the final formulation and the pH was adjusted to7.2–7.4 (measured at room temperature) using 1 N hydrochloric acid/1 NNaOH. The solution was made up to the final volume with water andsterile-filtered through a 0.2 μm filter. It was then filled into 5 mlinjection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength polysorbate 20(TWEEN® 20) stock solution was added, so that a concentration of 10μg/ml results in the finished formulation.

In each case 5 samples were then stressed in the rotation test and theturbidity was determined after various periods of time. The results areshown in the following table.

Number of test samples with turbidity >18 FNU after: Description 0 h 8 h16 h 24 h 32 h 40 h HMR1964 without addition 0 5 — — — — HMR1964 + 10μg/ml of 0 0 0 0 5 — TWEEN ® 20

The addition of polysorbate 20 delayed the occurrence of turbidity verymarkedly.

Example 3

Stabilization of HMR1964 by Addition of Poloxamer in the Rotation Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution which contained 4.5 mg/ml of phenol, 5 mg/ml of NaCl and 6mg/ml of trometamol in the final formulation and the pH was adjusted to7.2–7.4 (measured at room temperature) using 1 N hydrochloric acid/1 NNaOH. The solution was made up to the final volume with water andsterile-filtered through a 0.2 μm filter. It was then filled into 5 mlinjection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength poloxamer 171 (e.g.GENAPOL®) stock solution was added, such that a concentration of 10μg/ml results in the finished formulation.

In each case 5 samples were then stressed in the rotation test and theturbidity was determined after various periods of time. The results areshown in the following table.

Number of test samples with turbidity >18 FNU after: Description 0 h 8 h16 h 24 h 32 h 40 h HMR1964 without addition 0 5 — — — — HMR1964 + 0.01mg/ml of 0 0 0 2 5 — poloxamer 171

The addition of poloxamer 171 also delayed the occurrence of turbiditymarkedly and stabilizes the preparation.

Example 4

Stabilization of HMR1964 by Addition of Polysorbate 20 or Polysorbate 80in the Shaking Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution which contained 3.15 mg/ml of m-cresol, 5 mg/ml of NaCl and 6mg/ml of trometamol in the final formulation and the pH was adjusted to7.2–7.4 (measured at room temperature) using 1 N hydrochloric acid/1 NNaOH. The solution was made up to the final volume with water andsterile-filtered through a 0.2 μm filter. It was then filled into 5 mlinjection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength polysorbate 20(TWEEN® 20) stock solution was added, such that a concentration of 10μg/ml results in the finished formulation.

c) A further comparison solution was prepared identically as in b), butthis time polysorbate 80 (TWEEN® 80) was used instead of polysorbate 20.

The samples were shaken at 30° C. on a laboratory shaker (60 rpm) andthe turbidity of the samples was measured after specific times. Theresults are shown in the following table.

Shaking test, turbidity (FNU) after Addition Start 1 week 2 weeks 3weeks 4 weeks Without addition 0.55 2.04 4.86 6.12 10.51 0.01 mg/ml ofTween 20 1.75 2.60 2.44 2.44 3.80 0.01 mg/ml of Tween 80 2.38 2.98 2.863.01 4.14

Both the addition of polysorbate 20 and of polysorbate 80 had astabilizing effect on HMR1964 in the shaking test.

Example 5

Stabilization of HMR1964 by Addition of Zinc or Poloxamer (GENAPOL®) inthe Shaking Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution which contained 3.3 mg/ml of phenol, 5 mg/ml of NaCl and 6mg/ml of trometamol in the final formulation and the pH was adjusted to7.2–7.4 (measured at room temperature) using 1 N hydrochloric acid/1 NNaOH. The solution was made up to the final volume with water andsterile-filtered through a 0.2 μm filter. It was then filled into 5 mlinjection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength poloxamer 171(GENAPOL®) stock solution was added, such that a concentration of 10μg/ml results in the finished formulation.

c) A further comparison solution was prepared as described in a), butinstead of poloxamer, a corresponding amount of a 0.1% strength zincchloride stock solution was added to the solution before making up withwater, so that a concentration of 15 μg/ml of zinc results in thefinished formulation.

Shaking test, turbidity (FNU) after Addition Start 1 week 2 weeks 3weeks 4 weeks None 0.39 0.70 4.46 8.74 14.11 0.01 mg/ml of 0.36 0.570.52 1.59 0.89 poloxamer 0.015 mg/ml of Zn 1.02 0.68 0.70 0.56 0.86

Both an addition of zinc and the addition of poloxamer prevented theoccurrence of turbidity in the shaking test.

Example 6

Stabilization of HMR1964 by Addition of Poloxamer in the Rotation Test

a) Zinc-free HMR1964 (calculated such that a concentration of 3.5 mg/mlresults in the finished formulation) was dissolved in an aqueoussolution which contained 3.3 mg/ml of phenol, 5 mg/ml of NaCl and 6mg/ml of trometamol in the final formulation and the pH was adjusted to7.2–7.4 (measured at room temperature) using 1 N hydrochloric acid/1 NNaOH. The solution was made up to the final volume with water andsterile-filtered through a 0.2 μm filter. It was then filled into 5 mlinjection vials and sealed using caps.

b) A comparison solution was prepared identically, but before making upwith water a corresponding amount of a 0.1% strength poloxamer 171(GENAPOL®) stock solution was added, such that a concentration of 100μg/ml results in the finished formulation.

In each case 5 samples were then stressed in the rotation test and theturbidity was determined after various periods of time. The results areshown in the following table.

Number of test samples with turbidity >18 FNU after: Description 0 h 8 h16 h 24 h 32 h 40 h HMR1964 without addition 0 5 — — — — HMR1964 + 0.10mg/ml 0 0 0 0 1 5 of poloxamer 171

The addition of 100 μg/ml of poloxamer likewise stabilizes the HMR1964preparation very markedly.

1. An aqueous liquid formulation comprising at least one insulin; atleast one surfactant; optionally at least one preservative; andoptionally at least one of an isotonicizing agent, a buffer, and anexcipient, wherein the formulation is stable and free from or containsless than 0.2% by weight of zinc based on the insulin content of theformulation.
 2. The formulation as claimed in claim 1, wherein the atleast one surfactant is selected from at least one of alkali metalsoaps, amine soaps, alkaline earth metal soaps, alkylsulfates,alkylsulfonates, natural surfactants, cationic surfactants, fattyalcohols, fatty acids, esters of polyhydric alcohols, ethers ofpolyhydric alcohols, and polyols.
 3. The formulation as claimed in claim1, wherein the surfactant comprises a soap selected from at least one ofa stearate, a palmitate, an oleate, and a ricinoleate.
 4. Theformulation as claimed in claim 2, wherein the alkylsulfates areselected from at least one of sodium laurylsulfate, sodium cetylsulfate,and sodium stearylsulfate.
 5. The formulation as claimed in claim 2,wherein the natural surfactants are selected from at least one of bileacid salts, saponins, gum arabic, and lecithins.
 6. The formulation asclaimed in claim 2, wherein the cationic surfactants are selected fromat least one of alkonium halides, cetylpyridinium chloride, andalkyltrimethylammonium bromide.
 7. The formulation as claimed in claim2, wherein the fatty alcohols are selected from at least one of cetylalcohol, stearyl alcohol, and cholesterol.
 8. The formulation as claimedin claim 2, wherein the esters of polyhydric alcohols are selected fromat least one of esters of sorbitol, glycerol, sucrose, and polyethyleneglycol.
 9. The formulation as claimed in claim 2, wherein the esters ofpolyhydric alcohols are fatty acid esters.
 10. The formulation asclaimed in claim 2, wherein the ethers of polyhydric alcohols areselected from at least one of ethers of sorbitol, glycerol, sucrose, andpolyethylene glycol.
 11. The formulation as claimed in claim 1, whereinthe surfactant comprises at least one of a polysorbate, a sorbitanester, a polyoxyethylene stearate, a polyoxyethylene ether, and apolyethylene glycol ether.
 12. The formulation as claimed in claim 2,wherein the polyols are selected from at least one of polypropyleneglycols, polyethylene glycols, poloxamers, Pluronics, and Tetronics. 13.The formulation as claimed in claim 1, wherein the preservative isselected from at least one of phenol, cresol, chlorocresol, benzylalcohol, and parabens.
 14. The formulation as claimed in claim 1,wherein the isotonicizing agent is selected from at least one ofmannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, andglycerol.
 15. The formulation as claimed in claim 1, wherein theexcipient is selected from at least one of a buffer substance, an acid,an alkali, a salt, protamine, arginine, and amino quinurid.
 16. Theformulation as claimed in claim 15, wherein the buffer substance isselected from TRIS, phosphate, citrate, acetate, and glycylglycine. 17.The formulation as claimed in claim 1, in which the at least one insulinis present in a concentration of 60–6000 nmol/ml.
 18. The formulation asclaimed in claim 17, wherein the at least one insulin is present in aconcentration of 240–3000 nmol/ml.
 19. The formulation as claimed inclaim 1, in which the at least one surfactant is present in aconcentration of 0.1–10000 μg/ml.
 20. The formulation as claimed inclaim 19, in which the surfactant is present in a concentration of1–1000 μg/ml.
 21. The formulation as claimed in claim 1, in which thesurfactant and/or isotonicizing agent comprises at least one of glyceroland mannitol in a concentration of 100–250 mM.
 22. The formulation asclaimed in claim 21, further comprising chloride as a buffer substanceand/or isotonicizing agent in a concentration of up to 150 mM.
 23. Theformulation as claimed in claim 1, in which a buffer substance ispresent in a concentration of 5–250 mM.
 24. A process for the productionof the formulation as claimed in claim 1, wherein the components aremixed together in the form of aqueous solutions, the pH is adjusted to adesired level, and the mixture is made up to the final volume withwater.
 25. The formulation of claim 1 wherein the surfactant comprisespolysorbate 20 and the formulation further comprises cresol, trometamoland NaCl.
 26. An aqueous liquid formulation obtainable by the process asclaimed in claim
 24. 27. A method of treating diabetes mellituscomprising administering the formulation as claimed in claim 1 to apatient in need thereof.
 28. The formulation of claim 1 furthercomprising at least one insulin analog, insulin derivative, or activeinsulin metabolite.
 29. An aqueous liquid formulation comprising atleast one insulin; at least one surfactant; optionally at least onepreservative; and optionally at least one of an isotonicizing agent, abuffer, and an excipient, wherein the formulation is stable and freefrom zinc or contains less than a 2:6 ratio of zinc to insulin.
 30. Theformulation as claimed in claim 29, wherein the at least one surfactantis selected from at least one of alkali metal soaps, amine soaps,alkaline earth metal soaps, alkylsulfates, alkylsulfonates, naturalsurfactants, cationic surfactants, fatty alcohols, fatty acids, estersof polyhydric alcohols, ethers of polyhydric alcohols, and polyols. 31.The formulation as claimed in claim 29, wherein the surfactant comprisesa soap selected from at least one of a stearate, a palmitate, an oleate,and a ricinoleate.
 32. The formulation as claimed in claim 30, whereinthe alkylsulfates are selected from at least one of sodiumlaurylsulfate, sodium cetylsulfate, and sodium stearylsulfate.
 33. Theformulation as claimed in claim 30, wherein the natural surfactants areselected from at least one of bile acid salts, saponins, gum arabic, andlecithins.
 34. The formulation as claimed in claim 30, wherein thecationic surfactants are selected from at least one of alkonium halides,cetylpyridinium chloride, and alkyltrimethylammonium bromide.
 35. Theformulation as claimed in claim 30, wherein the fatty alcohols areselected from at least one of cetyl alcohol, stearyl alcohol, andcholesterol.
 36. The formulation as claimed in claim 30, wherein theesters of polyhydric alcohols are selected from at least one of estersof sorbitol, glycerol, sucrose, and polyethylene glycol.
 37. Theformulation as claimed in claim 30, wherein the esters of polyhydricalcohols are fatty acid esters.
 38. The formulation as claimed in claim30, wherein the ethers of polyhydric alcohols are selected from at leastone of ethers of sorbitol, glycerol, sucrose, and polyethylene glycol.39. The formulation as claimed in claim 29, wherein the surfactantcomprises at least one of a polysorbate, a sorbitan ester, apolyoxyethylene stearate, a polyoxyethylene ether, and a polyethyleneglycol ether.
 40. The formulation as claimed in claim 30, wherein thepolyols are selected from at least one of polypropylene glycols,polyethylene glycols, poloxamers, Pluronics, and Tetronics.
 41. Theformulation as claimed in claim 29, wherein the preservative is selectedfrom at least one of phenol, cresol, chlorocresol, benzyl alcohol, andparabens.
 42. The formulation as claimed in claim 29, wherein theisotonicizing agent is selected from at least one of mannitol, sorbitol,lactose, dextrose, trehalose, sodium chloride, and glycerol.
 43. Theformulation as claimed in claim 29, wherein the excipient is selectedfrom at least one of a buffer substance, an acid, an alkali, a salt,protamine, arginine, and amino quinurid.
 44. The formulation as claimedin claim 43, wherein the buffer substance is selected from TRIS,phosphate, citrate, acetate, and glycylglycine.
 45. The formulation asclaimed in claim 29, in which the at least one insulin is present in aconcentration of 60–6000 nmol/ml.
 46. The formulation as claimed inclaim 45, wherein the at least one insulin is present in a concentrationof 240–3000 nmol/ml.
 47. The formulation as claimed in claim 29, inwhich the at least one surfactant is present in a concentration of0.1–10000 μg/ml.
 48. The formulation as claimed in claim 47, in whichthe surfactant is present in a concentration of 1–1000 μg/ml.
 49. Theformulation as claimed in claim 29, in which the surfactant and/orisotonicizing agent comprises at least one of glycerol and mannitol in aconcentration of 100–250 mM.
 50. The formulation as claimed in claim 49,further comprising chloride as a buffer substance and/or isotonicizingagent in a concentration of up to 150 mM.
 51. The formulation as claimedin claim 29, in which a buffer substance is present in a concentrationof 5–250 mM.
 52. A process for the production of the formulation asclaimed in claim 29, wherein the components are mixed together in theform of aqueous solutions, the pH is adjusted to a desired level, andthe mixture is made up to the final volume with water.
 53. Theformulation of claim 29 wherein the surfactant comprises polysorbate 20and the formulation further comprises cresol, trometamol and NaCl. 54.An aqueous liquid formulation obtainable by the process as claimed inclaim
 52. 55. A method of treating diabetes mellitus comprisingadministering the formulation as claimed in claim 29 to a patient inneed thereof.
 56. The formulation of claim 29 further comprising atleast one insulin analog, insulin derivative, or active insulinmetabolite.